Methods for making a tissue engineered muscle repair (temr) construct in vitro for implantation in vivo

ABSTRACT

Provided herein are methods of culturing organized skeletal muscle tissue from precursor muscle cells by cyclically stretching and relaxing said muscle cells on a support in vitro for a time sufficient to produce said organized skeletal muscle tissue, including reseeding said organized skeletal muscle tissue by contacting additional precursor muscle cells to said organized skeletal muscle tissue on said solid support, and then repeating said step of cyclically stretching and relaxing said muscle cells in said support in vitro for time sufficient to enhance the density (i.e., increased number of nuclei and/or number of multinucleated cells) of said organized skeletal muscle tissue on said support.

RELATED APPLICATIONS

This application is a continuation application of PCT Application No.PCT/US2011/047600, filed Aug. 12, 2011, which in turn claims the benefitof U.S. Provisional Application No. 61/373,624, filed Aug. 13, 2010, thedisclosures of each of which are incorporated by reference herein intheir entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under contractW81XWH-09-1-0578 from the Department of Defense. The United StatesGovernment has certain rights to this invention.

FIELD OF THE INVENTION

The present invention concerns methods and apparatus for the growth ofskeletal muscle in vitro.

BACKGROUND OF THE INVENTION

Volumetric muscle loss due to a variety of causes, including congenitaland acquired conditions, invasive surgical procedures, and traumaticinjury, produces a physiological deficit for which there are currentlyno effective clinical treatments. Management involves the use ofexisting host tissue to construct muscular flaps or grafts. However,this approach is not always feasible, delaying the rehabilitationprocess and restoration of tissue function. The ability to createclinically relevant autologous tissue engineered muscle repair (TEMR)constructs in vitro for restoration of muscle mass and function in vivowould remove a major hurdle to the successful skeletal musclereconstructive procedures required to repair complex extremity andfacial injuries suffered by injured individuals. See, e.g., Yoo et al.,US Patent Application Publication No. US2006/0239981 (Oct. 26, 2006).

SUMMARY OF THE INVENTION

Provided herein are methods of culturing organized skeletal muscletissue from precursor muscle cells by cyclically stretching and relaxingsaid muscle cells on a support in vitro for a time sufficient to producesaid organized skeletal muscle tissue, including reseeding saidorganized skeletal muscle tissue by contacting additional precursormuscle cells to said organized skeletal muscle tissue on said solidsupport, and then repeating said step of cyclically stretching andrelaxing said muscle cells in said support in vitro for time sufficientto enhance the density (i.e., increased number of nuclei and number ofmultinucleated cells) of said organized skeletal muscle tissue on saidsupport.

In some embodiments, the reseeding step is carried out under staticconditions. In some embodiments, the reseeding step is carried out bycontacting a solution carrying precursor muscle cells to said organizedskeletal muscle tissue for a time of 10 minutes to two days.

In some embodiments, the reseeding step is carried out by contacting asolution carrying precursor muscle cells to said organized skeletalmuscle tissue inside a mold configured to confine a cell suspension ontop of one or more of the supports and/or supports seeded with cells.

In some embodiments, the cyclically stretching and relaxing said musclecells on a support in vitro comprises: (a) providing precursor musclecells on a support in a tissue media; then (b) cyclically stretching andrelaxing said support at least twice along a first axis during a firsttime period; and then (c) maintaining said support in a substantiallystatic position during a second time period; and then (d) repeatingsteps (b) and (c) for a number of times sufficient to enhance thefunctionality of the muscle tissue or produce organized skeletal muscletissue on said solid support from said precursor muscle cells (e.g.,increase the number of multinucleated cells, increase myotube width,enhance cellular alignment or orientation along an axis, etc.).

In some embodiments, the cyclically stretching and relaxing is carriedout at least three times (e.g., from 3 or 4 to 10 or 20 times) duringsaid first time period.

In some embodiments, the stretching comprises extending said support toa dimension between 5% and 15% greater in length than said staticposition. In some embodiments, the stretching comprises extending saidsupport to a dimension of between 8% and 12% greater in length than saidstatic position.

In some embodiments, the first time period is from 2 to 10 minutes induration; and wherein said second time period is from 5 to 40 minutes induration. In some embodiments, the repeating of steps (b) and (c) iscarried out for a time of five days to three weeks. In some embodiments,the reseeding step is repeated one, two, three, four or five or moretimes.

Multi-layered skeletal muscle tissues produced by the processesdisclosed herein are also provided. In some embodiments, the tissuecomprises elongated multi-nucleated muscle fibers or cells (e.g., from5, ‘0 or 15 to 100, 200, or 400 multinucleated cells per squaremillimeter of tissue or support surface area). In some embodiments, thetissue expresses acetylcholine (ACh) receptors (e.g., aggregated AChreceptors). In some embodiments, the tissue is suturable. In someembodiments, the construct further comprises or includes activatedsatellite cells or myoblasts.

Also provided are methods of treating a skeletal muscle injury in apatient in need thereof comprising grafting a skeletal muscle tissue(e.g., produced by the processes disclosed herein) into said patient ina treatment-effective configuration. Further provided is the use of askeletal muscle tissue as described herein for treating a skeletalmuscle injury.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Multiple seeding protocol of bladder acellular matrix (BAM)scaffold for TEMR. Scaffolds are initially seeded and incubated understatic conditions for ten days (A, B, & C). The seeded scaffolds arethen placed in the bioreactor for three days of pre-conditioning (D).Then, silicon molds are placed in the bioreactor (E), the myogenic mediais removed from the bioreactor, and the scaffolds are seeded a secondtime (F). After 6 to 24 hours of static incubation, myogenic media isrestored to the bioreactor wherein the scaffolds resume conditioning(G).

FIG. 2. Actin and desmin protein expression by TEMR constructs followingstatic and bioreactor preconditioning at 10% strain and 3stretch-relaxation cycles per minute for the first 5 minutes of everyhour (1×) or half hour (2×). Actin and nuclei were detected withphalloidin Alex 594 (red) and dapi (blue), respectively, while desminwas detected with goat anti-rat polyclonal primary antibody (1:50) andrabbit anti-goat fluorescein (green) secondary antibody (1:500). Allimages were captured at 400×; Scale bar=50 μm.

FIG. 3. Typical bioreactor preconditioning standard operating procedure(SOP).

FIG. 4. Triple immunostaining method used for discriminatoryvisualization of the impact of bioreactor preconditioning andmultiple/repeated cell seeding on the TEMR construct. Shown arerepresentative images of the morphological characteristics observedfollowing a 7d double-seeding bioreactor pre-conditioning protocol. Thecytosol of cells in the second seeding event was loaded with eithergreen (Cm-DiO) or red (Cm-DiI) fluorescent dye. Actin and nuclei of allcells were detected with phalloidin Alexa 594 (Red) or 488 (green) andDAPI (blue), respectively. During bioreactor preconditioning, constructswere stretched 3 times per minute for the first 5 minutes of every hour(A-F; 1×) or every half-hour (G-I; 2×) with a strain of 10%. Scalebar=50 μm.

FIG. 5. Effect of varying mechanical properties on scaffold TEMRconstruct morphology. All constructs were either static (A) or underwentuniaxial stretching in the bioreactor (B-D) for seven days.Bioreactor-preconditioned scaffolds were stretched three times perminute for the first five minutes of each hour (1×; B) or half hour (2×;C & D) with 10% (B & C) or 15% (D) strain. Immunofluorescence images areat 400× magnification with actin and nuclei stained red (phalloidin) andblue (dapi), respectively. Scale bar=50 um.

FIG. 6. Tissue engineered skeletal muscle construct morphology followingbioreactor preconditioning. Constructs either remained static for theduration of bioreactor preconditioning protocol (A), or were conditionedwith different bioreactor protocols (B-D). Scaffolds were uniaxiallystretched 3 times per minute for the first 5 minutes of every hour (B;lx) or half hour (C; 2×) for one week. Additionally, 2× constructsunderwent a second cell seeding, while in the bioreactor, three daysinto the one-week protocol and then resumed stretching following a 6hour static interval (D; 2×-DS). Constructs are stained for actin(phalloidin; green) and nuclei (dapi; blue). All images are at 400×magnification. The number of nuclei and number of multinucleated cellswere counted from the number of 400× images listed in parentheses thatwere derived from at least three different constructs. The number ofnuclei were counted using ImageJ software. The number of multinucleatedcells were determined by a blinded researcher. *Indicates that 2×-DSconstructs had significantly more nuclei (E) and multinucleated cells(F) than all other construct types (p<0.01). Values listed are means±SD.

FIG. 7. Acetylcholine receptor expression in TEMR constructs. For imagesA-C, actin, acetylcholine receptors, and nuclei were detected withphalloidin Alexa 594 (red), a-bungarotoxin Alexa 488 (green) and DAPI(blue), respectively. Image C represents the merger of images A & B. Forimage D, desmin was detected with a combination of goat antiratpolyclonal primary antibody (1:50) and rabbit anti-goat rhodamine (red)secondary antibody (1:500), and acetylcholine receptors and nuclei werestained with a-bungarotoxin Alexa 488 (green) and DAPI (blue),respectively. All images were captured at 400× magnification; Scalebar=50 μm.

FIG. 8. Rat muscle progenitor cell (MPC) protein expression and bladderacellular matrix scaffold characteristics. MPCs from primary culturewere passaged once, seeded on non-coated chamber slides, and thencultured for 1 day in proliferation media (See methods described inExample 3). Per protein marker, the total number of nuclei andpositively stained nuclei were counted in at least 12 high-powered field(400×) images from at least two different chamber slides. Over 800nuclei were counted for each protein marker with the number of positivecells expressed as percentage of total nuclei (D). Bladder acellularmatrix collagen scaffolds were cut to ˜3×1 sheet prior to implantation(E; scaffold was rehydrated in pink media for picture contrast). Young'smodulus was determined for seven sterilized and rehydrated scaffolds.Scaffolds were confirmed to be decellularized via the absence of aprotein signature (Ponceau) or specifically Gapdh (blot; G), as well asthe absence of nuclei (dapi; H). Protein expression (G) as well asnuclear staining via dapi is demonstrated on BAM scaffold following theaddition of MPCs.

FIG. 9. Cellular morphology and protein expression characteristics ofBAM-supported TEMR constructs developed under varying cultureconditions. Cell morphology of TEMR constructs generated under“Proliferation”, “Differentiation”, or “Mixed” culture conditions (SeeMethods in Example 3) are depicted in A, B, & C, respectively (400×images). For the generation of the “Mixed” construct group, a secondbatch of MPCs was added to an underlying layer of MPCs (i.e.,“Differentiation” constructs). To confirm adherence of the second MPCbatch, these cells were loaded with cytoplasmic fluorescent dye (Cm-Dio)and then visualized following preconditioning (D & E). The number ofnuclei (F) and the number of multinucleated cells (G) were quantitatedfor each construct type (See methods, * ≠Proliferation;#≠Differentiation, p<0.05). Muscle-specific protein expression of TEMRconstructs was characterized via Western blot (H). The optical densitiesof specified proteins were normalized to that of Gapdh for statisticalcomparisons among groups (I; * Significantly different fromProliferation, p<0.05). Protein expression of each construct type issummarized (J).

FIG. 10. LD muscle in vitro isometric force recovery following VMLinjury is dependent on TEMR construct type. Uninjured and injured butnon-repaired (NR) or TEMR construct (3 types, Proliferation,Differentiation, and Mixed)-repaired LD muscles were tested using directmuscle stimulation at 35° C. in an organ bath (See Methods in Example3). Isometric force as a function of stimulation frequency was assessedfor all experimental conditions at either one (A) or two (B) monthspost-injury. Force-frequency curves were fit with a Hill equation asdescribed in the methods. Peak isometric tetanic force functionaldeficits relative to the uninjured group mean was calculated for allexperimental groups at one (C) and two (D) months. For each post-injurytime, * ≠to NR while #≠to all other groups (p<0.05). Values areexpressed as means±SE. Sample sizes for each group at each post-injurytime are listed in Table 1.

FIG. 11. LD muscle tissue morphology after VML injury and immediaterepair with TEMR constructs. VML injured LD muscles that were either notrepaired (A & E) or repaired with “proliferation” (B & F),“differentiation” (C & G), or “mixed” (D & H) TEMR constructs wereretrieved one (A-D) and two (E-H) months post-injury and stained usingMasson's Trichrome (Red=tissue, Blue=Collagen, Black=Nuclei). * Markerof area of initial injury (A & E) or presumptive BAM collagen deposition(B-E & F-H). Images are 200× magnification with the scale bar=50 μm.

FIG. 12. Functional protein expression in regenerating muscle fibers andputative neo-tissue two months after TEMR construct treatment of VMLinjured LD muscle. Images are representative of tissue regeneration andformation observed in all three types of TEMR construct-repaired LDmuscles. Masson's trichrome staining and immunohistochemical stainingfor functional proteins is illustrated at the interface between theremaining native tissue and the TEMR construct (A-E) as well as forindependent tissue formed in BAM scaffolding (F-J). Insets show negativecontrol staining for the primary antibody. Images are 400× magnificationwith the scale bar=50 μm.

FIG. 13. Presence of vascular and neural structures one month after TEMRconstruct treatment of VML injured LD muscle. Images are representativeof vascular (#) and neural (*) structures that were identified viacharacteristic morphology and were observed in all TEMR construct groupsone month post-injury. Images are 400× magnification; Scale bar=50 μm.

FIG. 14. LD muscle protein expression two months post-injury. A) LDmuscles were probed for Pax7, desmin, myosin (MF20), junctophilin 1(JP1), and gapdh using SDS-PAGE and Western blotting (See methods inExample 3). B-D) Optical density was determined for each band andnormalized to gapdh. *, Significantly different from uninjured; #,Significantly different from all other groups (p<0.05). Values areexpressed as means±SE. Sample sizes for each group are listed inparentheses in panel D.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Provided herein and further described below are compositions and methodsuseful for producing functional muscle tissue in vitro for implantationin vivo. The disclosures of all United States patent references citedherein are hereby incorporated by reference to the extent they areconsistent with the disclosure set forth herein.

As used herein in the description of the invention and the appendedclaims, the singular forms “a,” “an” and “the” are intended to includethe plural forms as well, unless the context clearly indicatesotherwise. Furthermore, the terms “about” and “approximately” as usedherein when referring to a measurable value such as an amount of acompound, dose, time, temperature, and the like, is meant to encompassvariations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specifiedamount. Also, as used herein, “and/or” or “I” refers to and encompassesany and all possible combinations of one or more of the associatedlisted items, as well as the lack of combinations when interpreted inthe alternative (“or”).

“Implant” refers to a product configured to repair, augment or replace(at least a portion of) a natural tissue of a subject (e.g., forveterinary or medical (human) applications). The term “implantable”means the device can be inserted, embedded, grafted or otherwisechronically attached or placed on or in a patient. Implants include asupport having cells seeded thereon and/or subjected to bioconditioningaccording to some embodiments as described herein.

“Subjects” are generally human subjects and include, but are not limitedto, “patients.” The subjects may be male or female and may be of anyrace or ethnicity, including, but not limited to, Caucasian,African-American, African, Asian, Hispanic, Indian, etc. The subjectsmay be of any age, including newborn, neonate, infant, child,adolescent, adult and geriatric subjects.

Subjects may also include animal subjects, particularly vertebratesubjects, e.g., mammalian subject such as canines, felines, bovines,caprines, equines, ovines, porcines, rodents (e.g., rats and mice),lagomorphs, non-human primates, etc., or fish or avian subjects, for,e.g., veterinary medicine and/or research or laboratory purposes.

“Treat” refers to any type of treatment that imparts a benefit to asubject, e.g., a patient afflicted with or at risk for developing adisease (e.g., a musculoskeletal disease). Treating includes actionstaken and actions refrained from being taken for the purpose ofimproving the condition of the patient (e.g., the relief of one or moresymptoms), delay in the onset or progression of the disease, etc. Insome embodiments, treating includes reconstructing skeletal muscletissue (e.g., where such tissue has been damaged or lost by, e.g.,injury or disease) by implanting an anisotropic scaffold (with orwithout muscle cells) into a subject in need thereof. Scaffolds may beimplanted, e.g., at or adjacent to the site of injury, and/or at anothersite in the body of a subject that would impart a benefit to thesubject, as would be appreciated by one of skill in the art.

“Isolated” as used herein signifies that the cells are placed intoconditions other than their natural environment. Tissue or cells are“harvested” when initially isolated from a subject, e.g., a primaryexplant.

Muscle cells used to carry out the present invention are preferablymammalian muscle cells, including primate muscle cells, including butnot limited to human, pig, goat, horse, mouse, rat, monkey, baboon, etc.In general, such cells are skeletal muscle cells. Muscle cells of otherspecies, including birds, fish, reptiles, and amphibians, may also beused if so desired. In some embodiments the cells are precursor cells,or cells that are capable of differentiating into mature,multi-nucleates muscle cells, specifically skeletal muscle cells, underappropriate culture conditions and stimuli as described herein. Muscleprecursor cells are known. See, e.g., U.S. Pat. No. 6,592,623.

“Skeletal muscle cells” include, but are not limited to, myoblasts,satellite cells and myotubes. “Myoblasts” are a type of muscle precursorcell, and are normally closely associated with myofibers during thecourse of their life cycle in the vertebrate organism. If the myofiberis injured, the myoblasts are capable of dividing and repopulating it.Typically, after muscle injuries myofibers become necrotic and areremoved by macrophages (Hurme et al. (1991) Healing of skeletal muscleinjury: an ultrastructural and immunohistochemical study, Med. SciSports Exerc. 23, 801-810). This induces proliferation and fusion ofmyoblasts to form multinucleated and elongated myotubes, whichself-assemble to form a more organized structure, namely muscle fibers(Campion (1984) The muscle satellite cell: a review, Int. Rev. Cytol.87, 225-251). “Myotubes” are elongated, multinucleated cells, normallyformed by the fusion of myoblasts. Myotubes can develop into maturemuscle fibers, which have peripherally-located nuclei and myofibrils intheir cytoplasm (e.g., as found in mammals).

Cells may be syngeneic (i.e., genetically identical or closely related,so as to minimize tissue transplant rejection), allogeneic (i.e., from anon-genetically identical member of the same species) or xenogeneic(i.e., from a member of a different species). Syngeneic cells includethose that are autogeneic or autologous (i.e., from the patient to betreated) and isogeneic (i.e., a genetically identical but differentsubject, e.g., from an identical twin). Cells may be obtained from,e.g., a donor (either living or cadaveric) or derived from anestablished cell strain or cell line. For example, cells may beharvested from a donor (e.g., a potential recipient of a bioscaffoldgraft) using standard biopsy techniques known in the art.

“Supports” on which muscle cells may be seeded and grown to producecultured muscle tissue of the present invention include any suitablesupport. See, e.g., U.S. Pat. Nos. 6,998,418; 6,485,723; 6,206,931;6,051,750; and 5,573,784. Supports may be formed from any suitablematerial, including but not limited to synthetic or natural polymers,other biopolymers, and combinations thereof. Examples include collagensupports or decellularized tissue supports (e.g., obtained from smoothmuscle or skeletal muscle, such as a decellularized mammalian (e.g.,porcine) bladder. If desired, an angiogenic compound such as VEGF can beseeded on or carried by the solid support to facilitate the formation ofvascular cells or vasculature in the muscle tissue. The supports may beof any suitable configuration, but in some embodiments comprise, consistof, or consist essentially of a generally flat planar portion. Thesupport may be of any suitable thickness, but in some embodiments are atleast 20, 30, 50 or 100 uM thick, up to 600, 800, or 1000 uM thick, ormore.

In some embodiments, supports may include a polymeric matrix (e.g.,collagen, a hydrogel, etc.). In preferred embodiments of the presentinvention, supports have mechanical integrity sufficient to withstandthe mechanical stimulation (e.g., cyclic loading) in a bioreactor toproduce the desired skeletal muscle tissues. For example, in someembodiments supports are able to withstand the cell seeding andpreferred bioreactor pre-conditioning protocols described herein for atleast 5, 10, 15, 17 or 20 days or more.

Any suitable culture media can be used to grow cells in the presentinvention, including medias comprising serum and other undefinedconstituents, defined medias, or combinations thereof, such as RPMI,DMEM, etc.

The “primary culture” is the first culture to become established afterseeding disaggregated cells or primary explants into a culture vessel.“Expanding” or “expansion” as used herein refers to an increase innumber of viable cells. Expanding may be accomplished by, e.g.,“growing” the cells through one or more cell cycles, wherein at least aportion of the cells divide to produce additional cells. “Growing” asused herein includes the culture of cells such that the cells remainviable, and may or may not include expansion and/or differentiation ofthe cells.

“Passaged in vitro” or “passaged” refers to the transfer or subcultureof a cell culture to a second culture vessel, usually implyingmechanical or enzymatic disaggregation, reseeding, and often divisioninto two or more daughter cultures, depending upon the rate ofproliferation. If the population is selected for a particular genotypeor phenotype, the culture becomes a “cell strain” upon subculture, i.e.,the culture is homogeneous and possesses desirable characteristics(e.g., the ability to express a certain protein or marker).

“Express” or “expression” of a protein or other biological marker meansthat a gene encoding the same of a precursor thereof is transcribed, andpreferably, translated. Typically, according to the present invention,expression of a coding region of a gene will result in production of theencoded polypeptide, such that the cell is “positive” for that proteinor other biological marker.

In some embodiments, cells are passaged once, twice, or three times. Instill other embodiments, cells are passaged more than 3 times prior touse. In some embodiments, cells are passaged 0-1, 0-2 or 0-3 times. Insome embodiments, cells are passaged 1-2, 1-3, or 1-4 or more times. Insome embodiments, cells are passaged 2-3 or 2-4 or more times. Infurther embodiments, cells are passaged 5, 8, 10, 12 or 15 or moretimes. The number of passages used may be selected by, e.g., therelative production of one or more muscle cell proteins and/or markersof interest measured in the cell population after each passage.

Any suitable bioreactor device can be used to carry out the presentinvention, including those described in Yoo et al., US PatentApplication Publication No. US2006/0239981 (Oct. 26, 2006) thedisclosure of which is incorporated by reference herein in its entirety.

In some embodiments, a “mold” is provided which is configured to fitwithin the bioreactor and also designed to confine a cell suspension ontop of and/or within one or more of the supports and/or supports seededwith cells. The mold may be made of a light-weight material (e.g.,silicone, with a total weight, e.g., of 1-5 grams) and preferably doesnot significantly damage the underlying cellular structures when placedonto the support and/or support seeded with cells.

Multiple cell seeding protocols (with or without additional bioreactorpreconditioning) are contemplated according to some embodiments. As anexample, if each additional cell seeding is carried out during a time ofbetween 2 and 4 days, the number of cell seedings according to someembodiments may be 2, 3, 4, 5, 6, 7, or 8 or more.

The length of stretching of the solid support according to someembodiments may be to a dimension at least 2, 5, 7 or 10% greater inlength than the static position, and in some embodiments preferably notgreater than 12, 15 or 20%, and the relaxing may comprise retracting thesupport to a dimension not greater in length than the static position.In some embodiments the “static position” may be intermediate betweenthe stretched and relaxed position, and in such cases the relaxing maycomprise retracting the support to a dimension at least 2, 5, 7 or 10%lesser in length than the static position.

The first time period, during which the stretching and relaxing occurs,may be of any suitable length, for example from 2 or 3 minutes up to 10,20 or 30 minutes in duration or more. The step of cyclically stretchingand relaxing is typically carried out at least two or three times duringthe first time period (e.g., from 2, 3 or 4 times, up to 10 or 20 times)

The second time period during which the support is maintained in astatic position, may be of any suitable duration. In some embodimentsthe second time period is shorter than the first time period, and may befrom 1 or 2 minutes in duration up to 10 or 20 minutes in duration. Inother embodiments the second time period is longer than the first timeperiod, and may be from 10 or 20 minutes in duration up to 40, 60 or 90minutes in duration, or more. In some embodiments, the second timeperiod is from 50 to 58 minutes in duration. In some embodiments, suchas where the first time period contains comparatively long intervalsbetween stretching and relaxing, the need for a second time period maybe obviated altogether.

In one preferred embodiment, the support is cyclically stretched andrelaxed during a first “active” time period to a dimension of 10%greater and lesser in length than the static dimension at a rate of 3cycles per minute for a total of five minutes, followed by anapproximately 25 minute or 55 minute “rest” second time period,continuously for 1 to 3 weeks of in vitro culture. In some embodiments,this improved protocol results in an increase in the number ofmultinucleated cells, thicker myotube width, better cellular alignment,etc., in the construct.

In some embodiments, this improved protocol of cyclic stretching andrelaxing and/or multiple cell seeding may result in an increase in thenumber of multinucleated cells, thicker myotube width, better cellularalignment or orientation along an axis, etc., in the construct (by,e.g., 10, 20, 50, 80 or 100%).

“Oriented” cells typically have one (or more) axis of orientation (e.g.,longitudinal axis), which may be in any desired direction within theregion of interest. It will be appreciated that “orienting” as usedherein may include partial or total orientation, so long as a sufficientincrease in organization is achieved to produce the effect or benefitintended for the particular implementation of the methods describedherein. For example, fibers and/or cells may be oriented along alongitudinal axis such that greater than 70, 80, 90, or 95% or more ofthe fibers and/or cells are at an angle of 50, 40, 30, 20, or 10 degreesor less from the reference axis in any direction.

In some embodiments, the construct is characterized by the expression ofacetylcholine (ACh) receptors, and in some embodiments the ACh receptorsare aggregated. In some embodiments, aggregated ACh receptors mayinclude those which approximate the characteristic pretzel shape of amotor endplate in innervated mature fibers in vivo.

Skeletal muscle tissue produced as described herein may be used in vitroto examine the pharmacological or toxicological properties of compoundsof interest (e.g., by adding the compound of interest to a culturemedium in which the tissue is immersed, and examining the histologicalor mechanical properties of the tissue as compared to a control tissue).

Skeletal muscle tissue (with or without support) produced by the methodsof the present invention is preferably “suturable” in that it hassufficient structural integrity to be surgically sutured or otherwisefastened at either end when implanted and thereafter develop tensionupon contraction.

Skeletal muscle tissue produced as described herein may be used for thereconstruction of damaged tissue in a patient, e.g., a patient with atraumatic injury of an arm or leg. Such tissue may be utilized on thesupport (which is also implanted) or removed from the support andimplanted into the subject. The skeletal muscle tissue may be implantedto “build” soft tissue (e.g., at the interface between an amputated limband a prosthetic device) or to reconstruct (partially or totally) adamaged muscle (e.g., a muscle of the face, hand, foot, arm, leg, backor trunk). The cultured skeletal muscle tissue preferably has, in someembodiments, a size or volume of at least 1, 2, or 3 or more cubiccentimeters (not counting the volume of the support if present), and/ora length of 1 cm to 50 cm, to provide sufficient tissue mass forimplantation in a patient (e.g., in association with an existing muscleof the patient) and reconstruction of a skeletal muscle involved in, forexample, movement of fingers.

For allogenic transplant into a patient, skeletal muscle as describedherein may be matched or tissue-typed in accordance with knowntechniques, and/or the subject may be administered immune suppressiveagents to combat tissue transplant rejection, also in accordance withknown techniques.

The present invention is explained in greater detail in the followingnon-limiting Examples.

Example 1 Improvements to Muscle Bioreactor Conditioning to Form MoreMature or “Native-Like” Skeletal Muscle Tissue

Development of a Methodology to Perform Multiple Cell Seeding on BAMScaffolds in the Bioreactor.

As depicted in FIG. 1 a-d, our previous seeding model involved pipettingcells onto a bladder acellular matrix (BAM) scaffold under staticconditions and then placing the muscle construct in the bioreactor forpreconditioning.

In order to perform a second seeding on BAM scaffold that has alreadyundergone a period of bioreactor pre-conditioning, a technique wasdeveloped to seed the scaffold while it remains in the bioreactor (FIG.1 e,f). The primary challenge to this approach is ensuring optimalscaffold coverage and ample opportunity for cellular adherence.Light-weight (˜2 gram) silicon molds were constructed to create a customseeding chamber within the bioreactor that constrains the muscleprogenitor cell suspension to the top of each individual muscleconstruct. The placement of the silicon molds on the periphery of themuscle constructs does not appear to damage underlying cellularstructures. Thus, the bioreactor has been adapted to accommodate cellseeding during preconditioning without the need to remove the scaffold(FIG. 1 e,f).

Immunofluorescent microscopy was used to visualize myoblast and myotubecell morphological features on the BAM scaffold following static orbioreactor preconditioning at two different stretching frequencies (FIG.2) in order to assess the enhanced bioreactor preconditioning on TEMRconstructs in vitro.

Standard Operating Procedure (SOP).

An SOP for an improved bioreactor preconditioning protocol thatincorporates a second cell-seeding event is outlined in FIG. 3.

Discrimination Between Cells of Different Seedings.

To be able to discriminate between the cells that were originallystatically seeded prior to bioreactor preconditioning and those thathave been subsequently seeded during bioreactor preconditioning, a redor green fluorescent cytoplasmic dye (CM-diI or CM-diO, respectively,Invitrogen) was applied to the cells that were subsequently incorporatedonto the BAM scaffold in a second seeding event. In tandem, theretrieved constructs are stained with myogenic muscle markers (e.g.,myoD & desmin) with a green fluorescent dye and nuclei with DAPI (blue).This approach permits the simultaneous assessment of the presence ofmyoblasts as well as the formation/maturation of myotubes. In addition,the methodology permits evaluation of the contribution of each cellularseeding round to tissue formation for the TEMR construct.

FIG. 4 illustrates the application of this methodology to a BAM scaffoldseeded in the bioreactor. Actin positive and cytosolic dye positivecells were visualized on the same BAM scaffold using thebioreactor-based seeding methodology illustrated in FIG. 3. Moreover,DAPI staining of nuclei is demonstrated in FIG. 4, indicating thatemployment of this triple staining technique may be used to discriminatebetween cells of the primary and secondary cell seedings.

Implementation of a Multiple Cell Seeding Protocol.

Our previous seeding model involved pipetting cells onto BAM scaffoldsunder static conditions and then placing the muscle construct in thebioreactor for seven days of uniaxial stretching as follows: three timesper minute for the first five minutes of each hour with 10% strain,continuously for seven days. To implement a second seeding event, westopped the bioreactor three days into preconditioning and thenperformed a second seeding of the scaffold.

On day 1, BAM scaffolds with both a top and bottom silicon seedingchamber were initially wet with PBS and allowed to incubate at 37° C.for a minimum of 2 hours. For the initial seeding, MPCs of P1 were splitand counted. The PBS was removed from the scaffolds and the cells, at adensity of 1 million per cm² were pipetted onto the scaffold in a‘Z-like’ fashion. The seeded scaffolds were placed into the incubatorfor approximately 30 minutes in which after a volume of 6 mL, enough tocover the scaffold, of seeding media was added into the dish. Thescaffold was incubated overnight.

On day 2, more MPCs from tissue culture dishes were split and counted.Once the cells were ready for seeding, the scaffolds were removed fromthe incubator and the media was removed from their dish. Each scaffoldwas flipped to allow seeding of the second side. Cells were againpipetted in a ‘Z-like’ fashion onto the scaffold at the same density of1 million per cm². The scaffolds were placed into the incubator at 37°C. for 30 minutes and then seeding media was added back into the dishenough to cover the top of the scaffold. The scaffolds were allowed toincubate for 2 more days with no disturbance.

The seeding media was removed from the dish and changed todifferentiation media on day 5. Each dish received enough media to coverthe top of the scaffold which was approximately 6 mL. This process ofchanging the media was repeated on day 8 and 10. At each of these timesthe media used was still differentiation media.

On day 11, the scaffolds were removed from their dishes and placed intoa bioreactor for stretching. Bioreactor seeding media was used and totalvolume within the bioreactor was approximately 150 mL.

Day 15 includes the double seeding. MPCs were split from tissue cultureplates and again counted. These cells were then centrifuged andresuspended in a DMEM serum free media at approximately 1 million cellsper mL. CM-DiI or CM-DiO was added at a concentration of 1:2000 and thesolution was allowed to incubate for 15 minutes at 37° C. The solutionwas centrifuged and resuspended in a PBS wash. The wash procedure wasrepeated a total of 3 times. After washing, the cells were finallyresuspended in normal seeding media at density of 2 million cells per 1mL. Also during this time, the scaffolds inside the bioreactor werebeing prepared for a second layer. The bioreactor was first removed fromthe incubator and the media removed. Silicone seeding chambers werefitted both below and above each scaffold while still inside thebioreactor to allow containment of the cells on top of each scaffold.The colored cells were pipetted on to each scaffold at a density of 1million per cm² and allowed to sit undisturbed for 20 minutes. Afterthis time approximately 75 mL of bioreactor seeding media was added tothe bioreactor and it was placed inside the incubator for 6 hours. Afterthis time, the silicone chambers were removed from the top and bottom ofeach scaffold and 75 more mL of bioreactor seeding media were added intothe bioreactor. This procedure was not repeated for the following side;only one side of the scaffold received a second layer of cells.

Time Media Day 1 to Day 4 Seeding Day 5 to Day 11 Differentiation Day 12to Day 15 Bioreactor Seeding Day 15 Seeding (for cells)

Seeding Media DMEM (Low Glucose) 15% FBS 1% Antibiotic/Antimycotic (AA)Differentiation Media F-10 Media 2% HS 1% AA Bioreactor Seeding MediaDMEM (Low Glucose) 15% FBS 2% Antibiotic/Antimycotic (AA)

The constructs were kept in static culture for 6-24 hours in order tomaximize cellular adherence to the existing cell layer. Then, uniaxialstretching was resumed for the remainder of the preconditioning protocol(i.e., 2-3 days). Approximately 1 million cells per cm² were seeded ontothe scaffold during the seeding event. FIG. 4 shows representativeimages collected following the SOP described in FIG. 3 for theseexperiments. Note that in FIG. 4, green (CmDiO) and red (CmDiI)cytosolic dyes, respectively, were loaded into cells prior to the secondseeding only. A triple-staining immunofluorescence approach was used, inwhich the cytosol of cells used in the second round of seeding wasvisualized by loading of the cells with red or green dye, while theactin staining was visualized with phalloidin, and the nuclei of allcells on the scaffold were visualized via DAPI.

The major findings from these experiments were as follows:

-   -   Cells from the second seeding adhered to the scaffold or        pre-existing layer of cells;    -   Some of the added cells fused with the existing cells;    -   The scaffold was comprised of aligned, multi-nucleated,        actin-expressing cells; and    -   At the end of bioreactor preconditioning, a multilayer construct        was observed: That is, for example, 2, 3 or 5 layers of cells,        up to 6 or 8 layers of cells, are observed in at least portions        of the construct; while other portions of the construct may        remain a monolayer of cells.

Interestingly, the observed colocalization of cytosolic dye with actin(FIG. 4 last column), suggests that a portion of the cells in thesecond-seeding are fusing with the pre-existing layer of cells, creatinga population of elongated multi-nucleated myotubes. This is particularlyadvantageous for creation of TEMR constructs that better approximatemature muscle cell morphology and function, especially when oneconsiders that muscle cell maturation and hypertrophy may be limited bythe constraints of the nuclear domain.

Thus, increased fusing of the myoblasts into myotubes was observed withthe double seeding protocol (and not just more cells). This is evidencethat the cells are maturing more rapidly, proceeding down the pathway ofsatellite cell to myoblast to myotube, with the double seeding thanwithout, making it more likely that they will form functional myofiberswhen implanted in vivo.

Optimization of Bioreactor Preconditioning Mechanical Parameters (i.e.,Mechanical Stretch).

In addition to developing and implementing a bioreactor preconditioningprotocol that permits multiple/repeated cellular seeding, we alsocharacterized the morphology of TEMR constructs subjected to bioreactorpreconditioning using distinct stretching protocols, by varyingmechanical parameters. For these experiments, all TEMR constructs werekept in static culture for 10 days following initial cell seeding (FIG.2). Based on these data, it appears that bioreactor preconditioning at astrain of 10-15% and a frequency of 3 times per minute for the first 5minutes of every hour to half hour improves cell density,differentiation (e.g., multinucleated cells), alignment, and morphologycompared to constructs cultured statically for the same culture duration(See FIG. 5A for representative static culture image compared tobioreactor preconditioned constructs FIGS. 5B & 5C). Additionally,increasing the stretching frequency and applying a double seedingincreases the number of multinucleated cells on the scaffold (DSprotocol) (FIG. 6).

Expression of Acetylcholine Receptors.

During the characterization of TEMR construct morphology followingbioreactor preconditioning, we discovered that multinucleated cells ofTEMR constructs expressed acetylcholine (ACh) receptors and, in rarecases, exhibited aggregation of these receptors (FIG. 7). Interestingly,the aggregation of ACh receptors in one construct is beginning toexhibit the characteristic pretzel shape of a motor endplate in maturefibers (FIG. 7D). These novel findings are encouraging for thedevelopment of TEMR constructs that produce clinically relevant force,as innervation of implanted TEMR constructs is crucial for functionalrestoration of traumatically injured muscle tissue, and we now know thatthese cells 1) express ACh receptors and 2) have the capacity to developa motor endplate.

Example 2 Rodent Muscle Injury Model, Demonstrating that ImprovedBioreactor Protocols In Vitro Lead to Accelerated Maturation andImproved Functional Outcomes In Vivo

Experimental Design.

Different strategies of culturing a mixed population of muscle precursorcells (MPCs) on bladder acellular matrix (BAM) collagen scaffolds wereused to create tissue engineered muscle repair (TEMR) constructs withdistinctly different morphological and protein expressioncharacteristics. Briefly, one group of constructs experienced a shortcellular proliferation and growth period, a second group experienced aprolonged cellular maturation period, and a third group was designed toreflect both of these conditions by applying a second population of MPCsto an underlying layer of maturing cells three days before implantation.Based on the culture and seeding conditions used to generate eachconstruct type, the experimental groups are referred to as:Proliferation, Differentiation, and Mixed, respectively. TEMR constructsderived from all three in vitro procedures were implanted at the site ofa freshly made volumetric muscle loss injury in the latissimus dorsimuscle of nude mice. One and two months after injury and implantation,contralateral control, non-repaired, and TEMR construct-repaired (threegroups) LD muscles were retrieved. Subsequent assessments of functionalcapacity and tissue repair and regeneration were conducted to comparethe therapeutic benefits of these distinct TEMR constructs.

Animals.

Male Lewis rats (3-4 weeks) and female athymic nude/nude mice (8-10weeks) were used for muscle precursor cell donors or for in vivo studiesof VML injury repair, respectively. Rodents were purchased fromcommercial vendors (Harlan and Jackson Laboratories). All animalprocedures were approved by the Wake Forest University IACUC and are inaccordance with animal use guideline set by the American PhysiologicalSociety.

Experimental Methods

Precursor Cell Isolation.

Tibialis anterior and soleus muscles from 3 to 4 week old male Lewisrats were harvested for primary cell culture using methodology describedpreviously. (Machingal) Briefly, skeletal muscles were digested in 0.2%Collagenase (Worthington biochemicals, Lakewood, N.J.) solution preparedin low glucose Dulbecco's Modified Eagle Medium (DMEM) (Hyclone, Logan,Utah) for two hours 37° C. Muscle tissue fragments were plated ontotissue culture dishes coated with MATRIGEL (BD Biosciences, Bedford,Mass.) in myogenic medium containing: DMEM high glucose supplementedwith 20% Fetal Bovine Serum (FBS), 10% Horse Serum, 1% Chicken EmbryoExtract and 1% Antibiotic/Antimycotic (Hyclone; Logan, Utah). Cells werepassaged at ˜75% confluence, cultured in DMEM low glucose supplementedwith 15% Fetal Bovine Serum (FBS) and 1% Antibiotic/Antimycotic and usedfor seeding at the second passage.

BAM Preparation.

BAM scaffolds were prepared from porcine urinary bladder in accordancewith known techniques. (Machingal et al., 2011) Briefly, the bladder waswashed and trimmed to obtain the lamina propria, which was placed in0.05% Trypsin (Hyclone, Logan, Utah) for one hour at 37° C. The bladderwas then transferred to DMEM solution supplemented with 10% FBS and 1%Antibiotic/Antimycotic and kept overnight at 4° C. The preparation wasthen washed in a solution containing 1% Triton X (Sigma-Aldrich, StLouis, Mo.) and 0.1% Ammonium hydroxide (Fisher Scientific, Fairlown,N.J.) in de-ionized water for 4 days at 4° C. Finally, the bladder wasthen washed in de-ionized water for 3 days at 4° C. The absence ofcellular elements and preservation of structural components wasconfirmed by histological assessments. The decellularized scaffold wasfurther dissected to obtain a scaffold with of 0.2-0.4 mm thickness;dimensions suitable for implantation in the surgically created mouse LDdefect. The prepared acellular matrix was then cut into strips of 3 cm×2cm size and placed onto a custom designed seeding chamber made ofsilicon (McMaster Carr, Cleveland, Ohio). Scaffolds and silicon seedingchambers were then individually placed in six well culture dishes andsterilized by ethylene oxide (FIG. 9F).

TEMR Construct Preparation.

Sterilized scaffolds in custom-made silicon seeding chambers were keptimmersed in a seeding media consisting of DMEM solution supplementedwith 15% FBS and 1% antibiotic/antimycotic media for at least 12 hoursat 37° C. prior to seeding. MPC's (Passage 2) were then seeded at aconcentration of 1 million cells per cm², and after 12 hours, theseeding chamber was flipped and a concentration of 1 million cells percm² was seeded on the other side. After a total of three days in seedingmedia, the “Proliferation” group was collected for either in vitroanalyses or implantation.

Constructs belonging to the “Differentiation” or the “Mixed” groups werethen immersed in differentiation media (F12 DMEM, 2% horse serum, 1%AA), wherein the cells were cultured for an additional 7 days. After atotal of 10 days of static culture, the cell-seeded scaffolds (i.e.,tissue engineered skeletal muscle or TE-MR) were then placed in abioreactor system, in accordance with known techniques. The bioreactorsystem consisted of a computer-controlled linear motor powered actuatorthat directed cyclic unidirectional stretch and relaxation. To permitapplication of the cyclic stretch protocol, one end of the TE-MRconstruct was attached to a stationary bar, while the other end wasconnected to a movable bar attached to the actuator. TEMR constructswere subjected to ˜10% strain, 3 times per minute for the first fiveminutes of every hour, for five to seven days in accordance with knowntechniques. Constructs that underwent the full static and dynamicdifferentiation protocols comprised the “Differentiation” group.Additionally, a third set of constructs called the “Mixed” group wascreated by stopping uniaxial stretching midway through preconditioning(i.e., 2-3 days), applying a second set of MPCs (1^(st) or 2^(nd)passage) at a density of 1 million cells per cm² to only one side of theconstruct, allowing for static cellular adherence over a 6 to 12 hourperiod, and then proceeding with uniaxial stretching (same conditions)for two days. During the entire cell culture process, both cell seededsurfaces (i.e., top and bottom of the same BAM scaffold) were fullyimmersed in media, the constructs were continuously aerated with 95%air-5% CO₂ at 37° C. in an incubator and media was changed every 3 days.

Immunocytochemistry and Analysis.

MPCs (P2) were seeded either on uncoated chamber slides or BAM scaffoldsat a density of 1 million cells per cm². Whole mount staining wasperformed by fixing the cells in 2% formalin, washing in PBS-glycine (10mM), permeabilized with 0.5% triton, and then washed again inPBS-glycine. Cells were then blocked in 3% (w/v) non-fat dried milk inPBS for 30 minutes at room temperature prior to incubation with primaryantibodies (1:50 in PBS) raised in mouse against desmin (SantaCruz,-7955), myoD (Hybridoma Bank), and Pax7 (Hybridoma Bank) orphalloidin-Alexa Fluor 488 or 594 conjugated (1:50, Invitrogen) for onehour. Following washing in PBS, cells were incubated in TexasRed-conjugated anti-mouse IgG (Vector; 1:100) secondary antibody forthirty minutes and were then washed again in PBS. Probed specimens werethen coverslipped with ProLong Gold including DAPI (invitrogen-P36931).

To determine the percentage of P2MPCs expressing pax7, myoD, or desminon chamberslides, the total number of nuclei and positively labelednuclei were counted in at least 12 high-powered field (400×) images fromat least two different chamber slides, resulting in over 800 nucleicounted per protein marker. The percentage of positive cells areexpressed as total positive cells of total cells counted.

To assess the cellular morphology and number nuclei on BAM scaffolds,the number of nuclei and number of multinucleated cells were countedfrom 400× images derived from at least three different constructs each.The number of nuclei were counted using ImageJ software. The number ofmultinucleated cells was determined by a researcher who was blinded tothe experimental conditions. Multinucleated cells were defined as astructure in which two or more nuclei were associated with the same setof actin stress fibers.

BAM Scaffold Mechanical Testing.

Uniaxial tensile mechanical testing was performed using an Instron55401. Prepared sterilized BAM scaffolds were incubated at 37° C. inDMEM for ˜24 hours prior to testing. BAM samples were uniformly preparedwith a width of 3.75 mm using a standard steal press. Samples were kepthydrated during preparation and testing. Pretension was set to 0.2 N.Samples were tested to failure using a strain rate of 0.5 mm/s. Young'smodulus was calculated from the slope of the linear portion of thestress-strain curve using.

Surgical Creation of VML Injury and TEMR Construct Implantation.

VML injury was created by surgically creating a critical size defect ofthe LD muscle in anesthetized (isoflurane) nu/nu mice using similarmethodology to our previous report. (Machingal) A longitudinal incisionwas made along the midline of the back. The trapezius muscle that coversthe LD muscle was lifted to expose the LD muscle without removing thetendon inserted at the humerus. Suture markers were then placed on theLD muscle demarking the superior half of the spinal fascia and themedial half of the of the muscle head at the humerus. The medial half ofthe muscle was then excised using a fine scissor. Using thismethodology, a defect weighing ˜18 mg was excised from the LD muscle.The injured LD muscle was then either left without further treatment oran ˜3×1 cm TEMR construct was sutured (Vicryl 6-0) to the site ofinjury. In all cases, the fascia and skin were then sutured closed andthe animals were allowed to recover from anesthesia.

In Vitro Functional Assessment.

Whole LD muscles were dissected free and studied in vitro using a DMTorgan bath system (DMT Model 750TOBS) and similar methodology inaccordance with known techniques. LD muscles were mounted in an organbath chamber containing a Krebs-Ringer bicarbonate buffer (pH 7.4) with(in mM) 121.0 NaCl, 5.0 KCl, 0.5 MgCl₂, 1.8 CaCl₂, 24.0 NaHCO₃, 0.4NaH₂PO₄, and 5.5 glucose (the buffer was equilibrated with 95% O₂-5% CO₂gas). The distal tendon was attached by silk suture and cyanoacrylateadhesive to a fixed support, and the proximal tendon was attached to thelever arm of a force transducer (DMT 750TOBS). The muscle was positionedbetween custom-made platinum electrodes. Direct muscle electricalstimulation (0.2 ms pulse at 30V) was applied across the LD muscle usinga Grass S88 stimulator (Grass Instruments, Quincy, Mass.). Real timedisplay and recording of all force measurements were performed on a PCwith Power Lab/8sp (ADInstruments, Colorado Springs, Colo.).

Once the LD muscles were mounted in the organ bath, the muscles wereallowed to equilibrate for 5 minutes prior to determining optimalphysiological muscle length (L_(o)) via a series of twitch contractions.Maximal force as a function of stimulation frequency (1-200 Hz) wasmeasured at 35° C. during isometric contractions (750 ms trains of 0.2ms pulses), with 2 min between contractions. Absolute forces (mN) as afunction of stimulation frequency were fit with the following equation:

f(x)=min+(max−min)/[1+(x/EC50)^(−n)].  Eqn 1.

Where x is the stimulation frequency, min and max are the smallest(i.e., twitch; P_(t)) and largest (i.e., peak tetanic; P_(o)) respectiveforces estimated. EC50 is the stimulation frequency at which half theamplitude of force (max−min) is reached and n is the coefficientdescribing the slope of the steep portion of the curve. Measured P_(t)and P_(o) and maximal tetanic force at 80 Hz (P_(80Hz)), an index ofmeasured force at approximately EC50, were compared during statisticalanalyses.

Additionally, P_(o) was normalized to an approximate physiologicalcross-sectional area (PCSA), which was calculated using the followingequation:

PCSA={wet wt (g)/[muscle density (g/cm³)*(muscle length (cm))]}  Eqn 2.

Where muscle density is 1.06 g/cm³. For a subset of muscles, followingforce-frequency testing a caffeine contracture force assessment wasperformed. For these studies, a maximal caffeine contracture responsewas elicited by exposing the muscle to 50 mM caffeine during twitchcontractions at a rate of 0.2 Hz. This concentration of caffeine waschosen because concentrations in the mM range have been previously shownto maximally stimulate whole uninjured and injured rodent skeletalmuscle. During this testing, resting tension of the muscle increasesuntil active force and resting tension are indistinguishable and thenthe response plateaus. Peak caffeine contracture force was defined asthe tension measured at this steady-state response.

Western Blotting.

TEMR constructs collected before implantation were rinsed with PBS andthen minced and incubated for 30 minutes in 200 μL of NP-40 lysis bufferwith a protease inhibitor cocktail (PIC: 40 μL/mL; Sigma P8340) restingon ice. Following incubation, the lysis suspension was centrifuged at7000 g for 10 minutes at 4° C. The supernatant was stored at −80° C.until further use.

Uninjured, injured, and injured and repaired whole LD muscles were snapfrozen in liquid nitrogen and stored at −80° C. LD muscles were thawedon ice, minced in 800 μL of homogenization buffer A (250 mM sucrose, 100mM KCl, 20 mM MOPs, 5 mM EDTA, pH 6.8)+PIC and then homogenized using aPowerGen 125 tissue homogenizer (Fisher Scientific) to make a wholemuscle homogenate. A portion (675 μL) of the whole homogenate was thenfurther processed to extract the myofibrillar fraction in accordancewith known techniques. Whole homogenates were centrifuged at 10,000 gfor 10 min at 4° C. The pellet was then resuspended in a 800 μL of washbuffer (175 mM KCl, 2 mM EDTA, 0.5% TritonX100, 20 mM MOPs, pH 6.8)prior to undergoing a second centrifugation at 10,000 g for 10 min at 4°C. The pellet was then resuspended in 500 μL of homogenization buffer C(150 mM KCl, 20 mM MOPs, pH 7.0) with protease inhibitor cocktail. Wholemuscle and myofibrillar fraction homogenates were stored at 80° C. untiluse. Protein concentration in homogenates was determined using aBradford assay (Biorad Protein Assay Dye Reagent—500-0006).

TEMR construct, whole muscle, and myofibrillar homogenates were dilutedin lamealli sample buffer with β-mercaptoethanol and then placed inboiling water for 3 minutes. From each respective homogenate type 60,25, and 15 μg of protein per sample was loaded into 7, 7, and 10%polyacrylamide gels and separated using SDS-PAGE. The separated proteinswere then transferred to a PVDF membrane (Millipore, Immobolin 0.45 μmpore), which was blocked overnight at 4° C. in 5% (w/v) non-fat driedmilk suspended in PBS-Tween. For the TEMR construct protein expressioncharacterization, membranes were probed with mouse-derived anti-desmin(Sigma D1033; 1:200), pax7 (Hybridoma Bank; 1:25), myosin (HybridomaBank MF20; 1:100), embryonic myosin heavy chain (Hybridoma Bank F1.652;1:100), and gapdh (Millipore MAB374; 1:1000) in PBS-T for three hours atroom temperature. For the whole LD muscle homogenate analysis, membraneswere probed with rabbit-derived anti-junctophilinl (Invitrogen 40-5100;1:20,000) and mouse-derived anti-desmin (Sigma D1033; 1:200), pax7(Hybridoma Bank; 1:50), and gapdh (Millipore MAB374; 1:1000) in PBS-Tfor two hours at room temperature. For the myofibrillar fractionhomogenate analysis, membranes were probed with mouse-derivedanti-myosin (Hybridoma Bank MF20; 1:500) and gaph (Millipore MAB374;1:1000). Following washing in PBS-T, membranes were incubated inanti-mouse or rabbit HRP conjugated secondary antibodies (Cell Signal7074 & 7076) in PBS-T (1:20,000) for two hours at room temperature.Membranes were washed in PBS-T before detection using a SuperSignal WestFemto Chemiluminescent Substrate kit (Thermo Scientific 34096) andFujifilm Intelligent Dark Box (LAS-3000). Optical density of the blotwas determined using ImageJ. All protein markers were normalized to theoptical density of gapdh.

Histology and Immunohistochemistry.

LD muscles from all experimental groups were fixed in 10% neutralbuffered formalin and stored in 60% ethanol. All samples were processed(ASP300S, Leica Microsystems, Bannockburn, Ill.) and then embedded inparaffin (EG1160, Leica Microsystems, Bannockburn, Ill.). Seven μM thickserial sections were cut from the paraffin embedded blocks and Masson'sTrichrome staining and immunohistochemical staining was performed usingstandard procedures. Immunohistochemical staining was performed usingantibodies to detect desmin (M0760, 1:75, Dako, Carpinteria, Calif.),junctophilin 1 (Jpl; Invitrogen 40-5100, 1:120), myosin (MF-20, 1:10),ryanodine receptor 1 (RyR1; 34C, 1:10), and Pax7 (1:150). MF-20, RyR1,and Pax7 antibodies were acquired from Developmental Studies HybridomaBank, Iowa City, Iowa. Biotinylated anti-mouse IgG (MKB-2225, 1:250,Vector Laboratories Inc, Bulingame, Calif.) and anti-rabbit (BA-1000,1:500, Vector Laboratories Inc, Bulingame, Calif.) secondary antibodieswere used to detect mouse (desmin, MF-20, RyR1, Pax7) and rabbit (Jpl)primary antibodies. The sections were next treated with Avidin BiotinComplex Reagent (PK-7100, Vector Laboratories Inc, Bulingame, Calif.)and then visualized using a NovaRED substrate kit (SK-4800, VectorLaboratories Inc, Bulingame, Calif.). Finally, the sections werecounterstained using Gill's Hematoxylin (GHS280, Sigma-Aldrich, St.Louis, Mo.). Tissue sections without primary antibody were used asnegative controls. Images were captured and digitized (DM4000B LeicaUpright Microscope, Leica Microsystems, Bannockburn, Ill.) at varyingmagnifications.

Results

BAM Characterization.

BAM scaffolds were prepared and seeded with MPCs as describedpreviously. (Machingal) A cellularity of the BAM scaffolds was confirmedby looking for the presence of nuclei or cellular protein (FIG. 1). Inboth cases, cellular presence was not observed, as nuclei were notpresent upon dapi whole mount staining and no cellular protein wasdetected via Bradford assay or visually on PVDF membranes followingSDS-PAGE. Tensile mechanical properties of BAM scaffolds prior toseeding with MPCs were characterized. BAM scaffolds exhibited a Young'sModulus of 7.62±1.25 MPa with a stress of 1.13±0.21 MPa at failure,which are similar values to other previously described acellularcollagen matrices.

Precursor Cell Characterization.

Cells from primary culture were passaged twice, seeded on glass chamberslides with no coating, and incubated for one day in proliferationmedia. At this time and under these conditions, the percentage of cellsexpressing Pax7, MyoD, and desmin was ˜36, 34, and 21% respectively(FIG. 8). This analysis performed on uncoated glass chamber slides mayunderestimate the percentage of the myogenic cell population on BAMscaffolds, which provide a more elastic surface than either glass orplastic.

TEMR Construct Characterization Prior to Implantation.

TEMR constructs generated under “Proliferation”, “Differentiation”, or“Mixed” culture conditions exhibited different morphologicalcharacteristics (FIG. 9). Under proliferation conditions MPCS appearedunfused and were not aligned (FIG. 9A), while under differentiationconditions (serum starvation+uniaxial mechanical strain) MPCs exhibitedan elongated and aligned morphology, however, the number ofmultinucleated cells was not significantly increased (FIG. 9G). Also,the total number of nuclei was significantly reduced followingdifferentiation culture conditions. The addition of a second batch ofMPCs to an underlying layer of differentiating cells (i.e., “Mixed”culture conditions) promoted an elongated and aligned cellularmorphology, a partial restoration of the total number of nuclei on thescaffold, and a significant increase in the number of multinucleatedcells. It is likely that the latter observation was achieved via fusionof a portion of the second batch of MPCs with the underlying cellularlayer.

Muscle protein characterization revealed that all construct typesexpressed both immature and mature muscle markers, suggesting that eachconstruct type is comprised of MPCs under multiple states ofdifferentiation and maturation. All constructs expressed similar Pax7and desmin protein expression relative to gapdh (FIG. 9I). However, TEMRconstructs generated under differentiation conditions expressedsignificantly less myosin and embryonic myosin heavy chain (MHC_(emb))relative to gapdh than under proliferation conditions. The addition ofMPCs under the mixed culture conditions promoted partial restoration ofmyosin and MHC_(emb).

In Vitro Isometric Strength Analysis.

Uninjured or injured LD muscles that received no repair (NR) or repairwith TEMR construct underwent in vitro isometric force-frequency testingeither one or two months post-injury. For NR muscles, peak isometrictwitch force (P_(t)), maximal isometric force at 80 Hz stimulation(P_(80Hz)), and peak tetanic force (P_(o)) were significantly reduced by˜66, 71, and 75% compared to uninjured values one month post-injury(FIG. 10; Table 1). At two months post-injury, the NR group exhibitedsimilar functional deficits for P_(t) and P_(80Hz) (˜77 & 75%,respectively). Although, some recovery of P_(o) from one to two monthspost-injury was observed for NR muscles (FIG. 10; Table 1), a continued˜67% functional deficit of P_(o) two months post-injury indicates acritical size defect was achieved in this study using this VML injurymodel.

Functional recovery mediated via TEMR construct repair was constructtype and time dependent. At one month post-injury, TEMR constructsgenerated under both proliferation and mixed culture conditionsexhibited improved LD function compared to NR values. For example, atthis time P_(t), P_(80Hz), and P_(o) were greater by ˜96, 106, 111% forproliferation and by ˜50 (p=0.276), 128, and 120% for mixed groups. Incontrast, P_(t), P_(80Hz), and P_(o) produced by the differentiationTEMR construct group were not significantly different from NR (FIG. 10;Table 1). Moreover, at this time the proliferation and mixed groupsproduced similar P_(o) to each other (FIG. 10; Table 1) and ˜36(p=0.075) and 41% greater P_(o) than the differentiation group. Lastly,NR, differentiation, and mixed groups exhibited a significantly leftwardshift in the force-frequency curve (i.e., EC₅₀<Uninjured; Table 1),while the proliferation group was similar to uninjured muscle.

TABLE 1 LD muscle morphological characteristics and in vitro isometricforce parameters One Month Post-Injury Uninjured NR Prolif Dif MixedSample Size 22 5 6 7 8 Muscle Characteristics Wet Weight (mg)  90.3 ±3.7 102.4 ± 11.8 166.1 ± 10.1^(a,b) 154.8 ± 14.1^(a,b) 162.0 ±14.6^(a,b) L_(o) (mm)  34.9 ± 0.8  33.3 ± 1.7  33.8 ± 1.4  36.2 ± 1.5 34.9 ± 1.4 Isometric Force Parameters P_(t meas,) mN  46.8 ± 3.5  16.1± 2.6^(a)  31.6 ± 2.7^(a,b)  19.8 ± 3.2^(a)  24.1 ± 5.4^(a)P_(80 Hz meas,) mN 212.2 ± 8.9  61.0 ± 8.3^(a) 125.5 ± 9.6^(a,b)  95.6 ±13.2^(a) 138.8 ± 14.6^(a,b) P_(o meas,) mN 374.4 ± 12.1  93.1 ± 17.3^(a)196.9 ± 13.0^(a,b) 144.4 ± 21.9^(a) 203.2 ± 19.7^(a,b,d) EC₅₀, Hz  78.7± 1.6  68.7 ± 4.4^(a)  76.2 ± 3.6  68.4 ± 4.4^(a)  68.5 ± 3.6^(a) ncoefficient  4.1 ± 0.2  3.6 ± 0.2  4.6 ± 0.6  3.6 ± 0.4  4.4 ± 0.5Specific P_(o meas,) N · cm²  15.6 ± 0.7  3.2 ± 0.5^(a)  4.2 ± 0.2^(a) 3.4 ± 0.7^(a)  4.9 ± 0.7^(a) Caffeine, mN 112.5 ± 6.3 — — — — TwoMonths Post-Injury ANOVA NR Prolif Dif Mixed (p) Sample Size 4 6 7 8Muscle Characteristics Wet Weight (mg)  86.6 ± 11.5 152.0 ± 14.3^(a,b)151.6 ± 23.5^(a,b) 152.8 ± 17.0^(a,b) <0.001 L_(o) (mm)  32.6 ± 0.8 31.0 ± 2.2  31.9 ± 1.3  32.1 ± 0.6 0.099 Isometric Force ParametersP_(t meas,) mN  10.6 ± 2.4^(a)  21.9 ± 4.9^(a)  20.0 ± 2.9^(a)  39.0 ±4.3^(b,)* <0.001 P_(80 Hz meas,) mN  54.2 ± 9.9^(a) 102.0 ± 16.4^(a,b)104.9 ± 10.7^(a,b) 135.5 ± 17.1^(a,b) <0.001 P_(o meas,) mN 123.3 ±19.1^(a,)* 197.2 ± 24.0^(a,b) 200.1 ± 16.3^(a,b*) 259.3 ±19.3^(a,b,c,d,)* <0.001 EC₅₀, Hz  87.0 ± 3.3*  81.9 ± 2.3  81.8 ± 15* 85.3 ± 4.2* <0.001 n coefficient  4.4 ± 0.3  6.1 ± 1.4  4.2 ± 0.5  4.5± 0.3 0.127 Specific P_(o meas,) N · cm²  5.1 ± 1.0^(a)  4.5 ± 0.8^(a) 4.7 ± 0.3^(a)  6.4 ± 1.0^(a) <0.001 Caffeine, mN  41.3 ± 3.7^(a)  83.7± 10.8^(a,b)  67.3 ± 5.4^(a,b)  86.6 ± 5.5^(a,b) <0.001 Values are means± SE. LD muscle sample sizes are for measured electrical stimulationforce parameters. L_(o) is the optimal muscle length coinciding withpeak twitch force. Measured isometric twitch (P_(t)), tetanic force at80 Hz (P_(80 Hz)), and peak tetanic (P_(o)) force were elicted usingdirect electrical stimulation (0.2 ms pulse width; 30 V). EC₅₀ is thestimulation frequency at which half of the rise in amplitude of forceoccurred. The n coefficient is the slope of the linear portion of theforce-frequency curves depicted in FIG. 3. Absolute P_(o) was normalizedby physiological cross-sectional area (see methods) to determinespecific force. Following force-frequency testing a subset of musclesperformed an isometric caffeine (50 mM) contracture test. Denotationsindicate statistically significantly different group means (p < 0.05):^(a)= Uninjured; ^(b)= NR at same post-injury time; ^(c)= Proliferationat same post-injury time; ^(d)= Differentiation at same post-injurytime; *= 1 month for same experimental group.

At two months post-injury, all TEMR construct groups producedsignificantly greater P_(80HZ) and P_(o) than NR, although only themixed group produced significantly greater P_(t) (FIG. 10; Table 1).However, the magnitude of functional recovery was TEMR constructdependent. For instance, P_(o) produced by proliferation,differentiation, and mixed groups was ˜60, 62, and 110% greater than NRat two months, with the mixed group producing greater P_(o) than bothproliferation and differentiation groups (e.g., ˜30%>Differentiation).Additionally, the time-course of functional recovery was TEMR constructdependent. From one to two months, the differentiation and mixed TEMRconstruct groups exhibited a ˜39 and 28% improvement in P_(o),respectively, however, the proliferation group showed no significantimprovement (i.e., 0.2%) over this time. Lastly, a the leftward shift inthe force-frequency curves observed at one month post-injury for NR,differentiation, and mixed groups was rectified at two months.

Absolute forces were normalized to estimated physiological crosssectional area to calculate specific force (N·cm⁻²). There were nosignificant differences among experimental groups at either one or twomonths post-injury and all experimental groups produced significantlyless specific P_(o) than uninjured muscles (Table 1). LD muscle wetweight was consistently greater for all TEMR construct groups at one andtwo months post-injury compared to uninjured and NR groups, while LDmuscle length was similar among all groups.

For a subset of muscles, peak caffeine contracture force was measuredtwo months post-injury. All experimental groups were significantlyreduced compared to uninjured controls (Table 1). However, all TEMRconstruct groups produced similar contracture forces to each other andgreater contracture force than the NR group. Specifically,proliferation, differentiation, and mixed groups produced ˜103, 63, and110% greater contracture force than NR, respectively.

Cell and Tissue Morphology.

Tissue and cell morphology of VML injured LD muscles with and withoutTEMR construct repair were qualitatively characterized. Specifically,the area of VML injury, where the implanted TEMR constructs interfacewith the remaining tissue was of specific interest and is illustrated inFIG. 11 for NR and TEMR construct repair groups at one and two monthspost-injury. In agreement with functional deficits observed, the NRgroup exhibited gross tissue disruption, marked by the presence of smallmuscle fibers, increased collagen deposition, and monocytes indicating acontinued immune response at one month post-injury. Two monthspost-injury, the NR muscle appears to have completed the innatedegenerative and regenerative response to the VML injury. At this time,the NR tissue shows little monocyte presence, improved muscle fiberorganization, and collagen and adipose deposition at the site of injury.

In all cases, TEMR construct implantation resulted in improved tissuemorphology compared to NR muscles. At one and two months post-injury themuscle fibers at the initial site of injury present qualitatively lesssigns of damage, disruption, and monocyte presence in TEMR constructrepaired versus NR tissue (FIG. 11). There were, however, distinctdifferences in tissue morphology among TEMR construct groups. Forexample, while the muscle fibers at the interface were eitherregenerated or repaired by one month post-injury for the proliferationgroup, the remaining BAM scaffold was mostly devoid of a cellularpresence (FIG. 11B). A similar morphology was also observed at twomonths post-injury with TEMR construct implantation generated underproliferation conditions (FIG. 11F). In contrast, a cellular presencewithin the scaffold was observed in both the differentiation and mixedconstruct groups. And, from one to two months there appeared to be anincrease in muscle tissue formation both at the site of injury (FIGS.11C & G) and within the scaffold independent of the primary muscletissue (FIGS. 11D & H). Lastly, we also observed a marked vascular andneural presence at the tissue-scaffold interface one month post-injurywhen TEMR constructs were implanted, however, there was no obviousdifference in the occurrence of these structures among construct types(FIG. 13). As illustrated, the neural and vascular structures wereassociated with regenerating muscle fibers in most cases.

Functional Protein Expression.

To determine if regenerating or newly formed muscles fibers were capableof contributing to functional recovery, TEMR construct-repaired LDmuscles retrieved two months post-injury were stained using IHC for ahost of key proteins required for force production and transmission. Twoareas of interest within the repaired LD muscles were identified: 1) Thearea of initial VML injury at the interface between TEMR constructs andthe remaining native tissue (FIG. 12A-E); And 2) at sites of tissueformation within BAM scaffolding independent from the interface (FIG.12F-J). For all construct groups, muscle fibers in each of these areasstained positively (determined by negative control and striatedappearance) for desmin, myosin, ryanodine receptor 1 (RyR1), andjunctophilin 1 (JP1).

The relative content of specific muscle proteins involved in forceproduction and transmission can be reduced in injured muscle leading tofunctional deficits. In this study, relative protein content of desmin,jp1, and myosin (normalized to gapdh) was quantified in whole ormyofibrillar (myosin only) protein homogenates from uninjured and VMLinjured LD muscles two months post-injury (FIG. 14). No differencesamong uninjured, NR, and TEMR construct groups were observed for eitherdesmin or myosin. In comparison to all other treatment groups, JP 1 waselevated for TEMR constructs generated under proliferation conditions,although it is unclear what the physiological relevance of this increaseis.

Pax7 Expression in TEMR Construct Repaired LD Muscle.

Lastly, while the muscle fibers at the interface appear to havecompleted or nearly completed the regenerative response two months afterinjury (FIGS. 11 & 12) in TEMR construct-repaired LD muscles, fiberslocalized to the scaffold often appear smaller in diameter, suggestingthat the regenerative response is not completed in this area (FIG.12G-J). Because Pax7 expression, a satellite cell marker, increases inregenerating muscle following injury, protein expression of thisregenerative marker was determined in whole LD muscle homogenates (FIG.14). In comparison to uninjured values, Pax7 expression wassignificantly elevated for all TEMR construct groups, but not for the NRgroup.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of culturing organized skeletalmuscle tissue from precursor muscle cells, comprising: cyclicallystretching and relaxing said precursor muscle cells on a support invitro for a time sufficient to produce said organized skeletal muscletissue; reseeding said organized skeletal muscle tissue by contactingadditional precursor muscle cells to said organized skeletal muscletissue on said solid support; and then repeating said step of cyclicallystretching and relaxing said muscle cells in said support in vitro fortime sufficient to enhance the density of said organized skeletal muscletissue on said support.
 2. The method of claim 1, wherein said reseedingstep is carried out under static conditions.
 3. The method of claim 2,wherein said reseeding step is carried out by contacting a solutioncarrying precursor muscle cells to said organized skeletal muscle tissuefor a time of 10 minutes to two days.
 4. The method of claim 1, whereinsaid reseeding step is carried out by contacting a solution carryingprecursor muscle cells to said organized skeletal muscle tissue inside amold configured to confine a cell suspension on top of one or more ofthe supports and/or supports seeded with cells.
 5. The method of claim1, wherein said cyclically stretching and relaxing said muscle cells ona support in vitro comprises: (a) providing precursor muscle cells on asupport in a tissue media; then (b) cyclically stretching and relaxingsaid support at least twice along a first axis during a first timeperiod; and then (c) maintaining said support in a substantially staticposition during a second time period; and then (d) repeating steps (b)and (c) for a number of times sufficient to enhance the functionality ofthe muscle tissue or produce organized skeletal muscle tissue on saidsolid support from said precursor muscle cells.
 6. The method of claim 5wherein said cyclically stretching and relaxing is carried out at leastthree times during said first time period.
 7. The method of claim 5,wherein said stretching comprises extending said support to a dimensionbetween 5% and 15% greater in length than said static position.
 8. Themethod of claim 5, wherein said first time period is from 2 to 10minutes in duration; and wherein said second time period is from 58 to50 minutes in duration.
 9. The method of claim 5, wherein said repeatingof steps (b) and (c) is carried out for a time of five days to threeweeks.
 10. The method of claim 5, wherein said reseeding step isrepeated at least one time.
 11. The method of claim 1, wherein saidsupport comprises a hydrogel.
 12. The method of claim 1, wherein saidsupport comprises collagen.
 13. The method of claim 1, wherein saidsupport is porous.
 14. The method of claim 1, wherein said support isfrom 20 μM to 1000 μM thick.
 15. A muscle construct comprising elongatedmulti-nucleated muscle cells or fibers in multilayer configuration on asupport.
 16. The muscle construct of claim 15 produced by the process ofclaim
 1. 17. The muscle construct of claim 15, wherein said constructcomprises from 5 to 400 multinucleated cells per square millimeter oftissue or support surface area.
 18. The muscle construct of claim 15,wherein said construct further comprises activated satellite cells ormyoblasts.
 19. The muscle construct of claim 15, wherein said musclecells or fibers express acetylcholine (ACh) receptors.
 20. The muscleconstruct of claim 15, wherein said muscle cells or fibers compriseaggregated ACh receptors.
 21. The muscle construct of claim 15, whereinsaid muscle cells or fibers comprise aggregated ACh receptors forming apretzel shape characteristic of motor end plates.
 22. The muscleconstruct of claim 15, wherein said construct is suturable.
 23. A methodof treating a skeletal muscle injury in a patient in need thereofcomprising grafting the muscle construct of claim 15 into said patientin a treatment-effective configuration.